Cancer is one of the most notorious health problems worldwide. This project aims to find effective cures for cancer by immunotherapy, namely to treat patients with anticancer antibodies or with therapeutic cancer vaccines that can provoke anticancer immunities, which is considered the ideal therapy for cancer due to its potentially high efficacy and low toxicity. In this regard, tumor-associated carbohydrate antigens (TACAs), the abnormal glycans expressed by tumors, are important molecular motifs and targets. However, one of the major issues for the majority of TACAs is their immune tolerance, i.e., they are poorly or nonimmunogenic, a problem that is frequently difficult to overcome by simply coupling TACAs to a carrier protein. On the other hand, the tolerated TACAs are often the relatively rich and important antigens on tumor cells, rendering them very attractive targets for cancer vaccine development. This project aims to establish a strategy to overcome the problem of immune tolerance to TACAs and use the strategy to design and develop new TACA-based immunotherapies for cancer. The basic concept of the proposed strategy is to immunize cancer patients with a vaccine derived from an unnatural analog of a TACA and then treat the patients with an unnatural sugar that can glycoengineer tumor cells to express the unnatural TACA analog in place of the natural TACA. The pre-stimulated immune system will subsequently recognize and destroy the glycoengineered tumors. It is anticipated that vaccines made of unnatural TACA analogs can easily provoke antitumor immunities. Previous studies of this project have proved the two prerequisites for the strategy, namely that unnatural TACA analogs are highly immunogenic to induce strong T cell dependent immune responses and that tumor cells can be effectively glycoengineered to express unnatural TACA analogs. This proposal will continue to test various glycoconjugates as vaccines for the new immunotherapeutic strategy, verify other related issues, and evaluate in vitro and in vivo the new immunotherapy for treating melanoma, breast and colon cancer. The specific aims of this proposal are: 1) to synthesize and study new conjugate vaccines with the unnatural TACA analogs linked to monophosphoryl lipid A and a peptidic T cell epitope; 2) to develop a method to quantitatively analyze the expression of unnatural TACA analogs on cells as a result of glycoengineering, and to evaluate in vitro the efficacy and the selectivity of various antibodies to target and kill glycoengineered tumor cells; and 3) to study and evaluate in vivo the efficacy of the new immunotherapy to cure cancer and prevent tumor metastasis based on antibody and vaccine treatments. It is expected that through the proposed studies promising cancer immunotherapies can be identified for clinical trials and, ultimately, be developed into useful therapies for melanoma and breast or colon cancer. Moreover, a solution to the immune tolerance problem may have a significant impact on cancer immunology and other related areas as well. PUBLIC HEALTH RELEVANCE: This research project aims to establish a new strategy for cancer immunotherapy, i.e., to treat tumors using anticancer antibodies or therapeutic cancer vaccines, which is considered the ideal cure for cancer. For this purpose, unnatural analogs of the special carbohydrate antigens expressed by cancer cells will be prepared and coupled to carrier molecules to create highly immunogenic vaccines that can be used to provoke specific immune responses or prepare specific antibodies against the unnatural antigens. The vaccinated animals will be then treated with an unnatural sugar which can induce the expression of the unnatural antigens on cancer cells. Subsequently, the provoked immune system or the specific antibodies will recognize and eliminate the labeled tumors. The specific aims of this proposal are to identify the effective vaccines for immunization of animals (or patients) and the appropriate conditions for labeling of tumor cells and to verify the efficacies of the new immunotherapies to cure cancer.